Expression system

ABSTRACT

The present invention provides an expression system for producing a target protein in a host cell comprising a homologously integrated gene encoding T7 RNA polymerase, and a nonintegrated gene encoding a target protein.

The present invention relates to a novel host cell useful in anexpression system for producing target proteins.

Many expression systems are available for the purpose of producingtarget proteins in bacterial host cells. Many of these systems arederived from naturally occurring endogenous regulatory systems like thelactose (lac) and tryptophan (trp) operons of E. coli. There are alsoseveral systems that utilize components of phage expression regulatorynetworks like the Lambda promoter (P_(L)) system of Lambda phage.

However, among the most widely and routinely used systems for expressionof recombinant target proteins in E. coli at the laboratory level is abacteriophage T7 expression system. The expression system iscommercially available from Novagen, Inc. (Madison Wis.) and isdescribed in U.S. Pat. No. 4,952,496. The expression system comprises ahost cell comprising an integrated phage lysogen. The host cell is thentransformed with a nonintegrated gene under control of a phage promoter,wherein the nonintegrated gene encodes a target protein of choice.

Lambda DE3 lysogen is a recombinant phage that carries a clone of T7 RNApolymerase under control of lacUV5 promoter. Lambda DE3 lysogens areprepared by co-infecting a host cell with a Lambda DE3 phage lysate, ahelper phage lysate, and a selection phage lysate. The result of theco-infection is a host cell that has the Lambda DE3 phage incorporatedinto the host cells' chromosome. Although the Lambda DE3 phage isintegrated into the host chromosome at the Lambda integration site, theLambda DE3 phage is defective in its ability to be lytic. Thus, the DE3lysogen should be stable and should not subsequently lyse the cells toproduce infectious phage. Upon induction of the expression system, thehost cells make T7 RNA polymerase from the DE3 lysogen. The T7 RNApolymerase then binds the phage promoter of the nonintegrated targetgene and initiates synthesis of the target protein.

A T7 expression system provides many benefits that make it quitesuitable to express target proteins. For example, the T7 or T7lacpromoter of the target gene is a phage promoter that is unique to phageand is not recognized by the host cell RNA polymerases. Thus, expressionof the target protein is only initiated when T7 RNA polymerase ispresent. This helps to reduce the potential for expression of the targetprotein prior to induction. Expression of the target protein prior toinduction is not desirable because some target proteins have deleteriouseffects on host cell growth, thus, reducing maximum target proteinproduction.

Another example that makes the T7 expression system suitable to expresstarget proteins is the T7 promoter has been altered to include thelactose operator (lacO). The lacO is a binding site for the lactoseoperon repressor. The lactose repressor binds the lacO, which preventsthe T7 RNA polymerase from binding the T7lac promoter, thus effectivelyrepressing expression of the target protein. The repression isreversible upon addition of an inducing agent to the host cell. Theinducing agent knocks the lactose repressor off the lacO and allows theT7 RNA polymerase to bind the T7lac promoter and initiate expression ofthe target protein. Inclusion of lacO tightens the initiation ofexpression of the target protein by nearly 10-fold. This also helps toreduce the potential for expression of the target protein prior toinduction, which for some target proteins, have deleterious effects onhost cell growth, thus, reducing maximum target protein production. Thelactose repressor is produced from an endogenous host cell gene calledlacI. However, host strains with lacI gene cannot produce enough lactoserepressor to effectively repress expression of the target protein. Thus,to obtain the appropriate regulation of target protein the host strainshould also contain an extra lacI-gene or use an overexpressing hostcell comprising a lacI^(Q1) promoter.

Probably the single most advantageous characteristic of the expressionsystem is the fact the T7 RNA polymerase is nearly 12-fold moreprocessive than the host cell RNA polymerase. The high processivity ofthe T7 RNA polymerase can generate more than 60% of the cell's totalprotein as the target protein, making it among the most efficientexpression systems available.

The basis for the present invention, however, is the discovery that ininstances where the target protein is produced in large quantities,infectious phage is detectable in the fermentation broth. This suggeststhat the DE3 phage has regained its ability to be lytic. The high celldensities achieved during fermentation may be such that the infectiousphage is generated through low levels of recombination or illegitimaterecombination (reversion) resulting in excision of the lysogen.Nonetheless, regulatory agencies prohibit forward processing of afermentation broth that contains target proteins to be used aspharmaceuticals that have detectable levels of phage particles.

In light of this problem, the present invention provides an improved T7expression system. In the present invention, the T7 RNA polymerase geneis integrated into the chromosome of the host cell using a differentintegration mechanism. The present invention integrates a copy of the T7RNA polymerase gene into a nonessential site in the chromosome of thehost cell by homologous recombination instead of infecting the host cellwith defective phage. The host cell further comprises a nonintegratedgene encoding a target protein of choice. The integrated gene encodingthe T7 RNA polymerase is under control an endogenous regulatory systemof the host cell, while the nonintegrated gene encoding the targetprotein is under control of a phage regulatory system. When the hostcell is induced, a host cell RNA polymerase is able to bind to a hostcell promoter and initiate synthesis of the T7 RNA polymerase. The newlysynthesized T7 RNA polymerase is available to bind to a T7 or T7lacpromoter and initiate synthesis of the target protein. The result is aphage-free fermentation broth comprising the target protein.

The present invention provides a host cell comprising a homologouslyrecombinated T7 RNA polymerase gene under control of a lac promoterintegrated into the host chromosome. The T7 RNA polymerase is integratedinto the host cell chromosome without the use of a phage lysogen,resulting in no incorporation of additional phage DNA. Homologousrecombination can occur in any nonessential gene of choice, whereas thephage lysogen integrates only at sites driven by the infection process.The promoter can be the wild type lac promoter or a modified lacpromoter like, lacUV5.

The host cell can further comprise a nonintegrated gene encoding atarget protein wherein the nonintegrated gene is under control of a T7or T7lac promoter. Preferably the T7 promoter is T7lac. Preferably thetarget protein is parathyroid hormone (PTH) (1-84) or active fragmentsthereof, including N-terminal fragment 1-34, 1-31, 1-28, or analogs orderivatives thereof. In another embodiment, the target protein isglucagon-like peptide-1 (GLP-1), or analogs or derivatives thereof.

The present invention further provides an expression system forproducing phage-free fermentation broth comprising a target protein,wherein the expression system comprises a host cell with a homologouslyintegrated T7 RNA polymerase gene in a nonessential gene on a chromosomeof a host cell and a nonintegrated gene encoding the target protein.

The present invention further provides a process for preparing a hostcell comprising a homologously integrated T7 RNA polymerase gene. The T7RNA polymerase gene is integrated into any nonessential gene of the hostchromosome, preferably, the galactose operon of the host chromosome. TheT7 RNA polymerase gene can be integrated into the galactose operon froma plasmid selected from the group consisting of pHMM209, pHMM220,pHMM223, and pHMM228.

The present invention further provides a process for preparing a targetprotein which comprises expressing the target protein in a host cellcomprising a homologously integrated T7 RNA polymerase gene, and whereinthe target protein is phage-free. Preferably the target protein isparathyroid hormone (PTH) (1-84) or active fragments thereof, includingN-terminal fragment 1-34, 1-31, 1-28, or analogs or derivatives thereof.In another embodiment, the target protein is glucagon-like peptide-1(GLP-1), or analogs or derivatives thereof.

FIG. 1 shows a schematic representation of homologous recombination ofthe T7 RNA polymerase from the integration plasmid pHMM228 into the hostchromosome.

For purposes of the present invention, as disclosed and claimed herein,the following general molecular biology terms and abbreviations aredefined below. The terms and abbreviations used in this document havetheir normal meanings unless otherwise designated. Amino acidsabbreviations are as set forth in 37 C.F.R. § 1.822 (b)(2) (1994).

“Base pair” or “bp” as used herein refers to DNA. The abbreviationsA,C,G, and T correspond to the 5′-monophosphate forms of thedeoxyribonucleosides (deoxy)adenosine, (deoxy)cytidine,(deoxy)guanosine, and thymidine, respectively, when they occur in DNAmolecules. In double stranded DNA, base pair may refer to a partnershipof A with T or C with G. “Kilo-base” or “kb” refers to one thousand(1000) base pairs.

“Plasmid” refers to an extrachromosomal genetic element comprisingnucleic acid. Plasmids are generally designated by a lower case “p”followed by letters and/or numbers. The starting plasmids herein areeither commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accordance withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan. Plasmids comprise DNA molecules to which one or moreadditional DNA segments can or have been added. Some plasmids aretemperature sensitive while others are not. This means that atpermissive temperatures, some plasmids are self-replicating, and atnonpermissive temperatures, some plasmids are not self-replicating.

“Expression plasmid” as used herein refers to any nontemperaturesensitive plasmid in which a promoter to control transcription of theinserted DNA has been incorporated. A T7 expression plasmid comprises aT7 or T7lac promoter that controls expression of a target gene encodinga target protein. T7 expression plasmids are well known to theordinarily skilled artisan. T7 expression plasmids are commerciallyavailable from Novagen, Inc. (Madison Wis.), and include but are notlimited to the pET series of expression plasmids.

“Integration plasmid” as used herein refers to any temperature sensitiveplasmid in which a promoter to control transcription of the inserted DNAhas been incorporated. Additionally, the integration plasmid inserts aspecified segment of DNA into the chromosome of a cell. Integrationplasmids are derived from pMAK700 and pMAK705. The pMAK700 and pMAK705are generated as described by Hamilton, et al., J. Bacteriol.171:4617-4622, (1989) which is herein incorporated by reference in itsentirety. Integration plasmids of the present invention, pHMM228,pHMM209, pHMM220, and pHMM223 are described in detail below. Theseintegration plasmids comprise a lac promoter that controls expression ofthe T7 RNA polymerase gene encoding the T7 RNA polymerase.

“Transformation” refers to the introduction of a plasmid into anorganism so that the plasmid is replicable, either as anextrachromosomal element or by chromosomal integration. Methods oftransforming bacterial and eukaryotic hosts are well known in the art,many of which methods are summarized in J. Sambrook, et al., MolecularCloning: A Laboratory Manual, (1989). Successful transformation isgenerally recognized when any indication of the operation of thisplasmid occurs within the host cell. For example, a sensitive host cellwill become resistant to a selecting agent when the host cell istransfected with a plasmid that allows for the resistance.

“Permissive temperature” is the temperature which a plasmid aftertransformation into the host cell can self replicate independent of cellduplication. The permissive temperature as defined in this invention isa temperature typically less than 44° C., generally between about 20° C.and about 40° C., preferably between about 25° C. and 40° C., morepreferably between about 25° C. and 35° C., most preferably about 30° C.

“Nonpermissive temperature” is the temperature which a plasmid aftertransformation into the host cell cannot self replicate independent ofcell duplication. The nonpermissive temperature as defined in thisinvention is a temperature typically greater than 40° C., generallybetween about 40° C. and about 50° C., preferably about 44° C.

“Transcription” refers to the process whereby information contained in anucleotide sequence of DNA is transferred to a complementary RNAsequence by RNA polymerase. For example E. coli RNA polymerase transfersthe T7 RNA polymerase gene to the complementary RNA sequence which isthen translated into T7 RNA polymerase. Likewise, for example, T7 RNApolymerase transfers the target gene to the complementary RNA sequencewhich is then translated into the target protein.

“Translation” as used herein refers to the process whereby the geneticinformation of messenger RNA (mRNA) is used to specify and direct thesynthesis of a polypeptide chain.

“Isolated amino acid sequence” refers to any amino acid sequence,however, constructed or synthesized, which is locationally distinct fromthe naturally occurring sequence.

“Isolated DNA compound” refers to any DNA sequence, however constructedor synthesized, which is locationally distinct from its natural locationin genomic DNA.

“Promoter” refers to a DNA sequence which binds an RNA polymerase anddirects transcription of DNA to RNA. Example of promoters used hereinare lac, lacUV5, T7, T7lac, lacI^(Q1).

“PCR” refers to the widely-known polymerase chain reaction employing athermally-stable DNA polymerase.

“Primer” refers to a nucleic acid fragment which functions as aninitiating substrate for enzymatic or synthetic elongation in PCR.

“Parental cell” refers to a cell that is void of a lysogen and iscapable of self-replicating in vitro. The parental cell should also haveDNA sequences that are determinable and should be approximately 2 kb inlength of the host cell chromosome. These sequences should further be ina nonessential area of the cell. Preferably, the parental cell isbacterial. Preferably, the parental cell comprises DNA sequences of thegalactose operon or a segment thereof. Preferably, the parental cell isE. coli. Preferred E. coli parental cells are commercially availablefrom several suppliers such as Novagen, Inc. (Madison Wis.), and includebut are not limited to BL21, AD494, BLR, HMS174, Origami, and Tuner.

“Host cell” in the present invention refers to a parental cell thatcomprises a homologously integrated T7 RNA polymerase gene under controlof a lac promoter. The promoter can be the wild type lac promoter or amodified lac promoter like lacUV5. The host cell can further comprise anonintegrated gene under control of a T7 promoter. The promoter can bethe wild type T7 promoter or a modified T7 promoter like T7lac. Thenonintegrated gene encodes a target protein of choice. Upon induction ofthe host cell, T7 RNA polymerase is produced. The T7 RNA polymerase isthen available to produce the target protein in phage-free fermentationbroth.

“Phage-free” refers to no observable plaques on a lawn of bacteria whenincubated with fermentation broth. Assays used to test for phagecontamination are well known in the art.

“Homologously integrated gene” refers to a gene that is integrated intothe chromosome of a host cell by a method of homologous recombination.The method of homologous recombination proceeds between a DNA sequenceon the chromosome of the host cell and complementary sequences carriedon an integration plasmid that is present inside the cell aftertransformation. Preferably, the method of homologous recombination isperformed as taught by Hamilton, et al. in New method for generatingdeletions and gene replacements in Escherichia coli. J. Bacteriol.171:4617-4622, 1989, which is herein incorporated by reference.

“Complementary” as used herein, refers to pairs of bases (purines andpyrimidines) that associate through hydrogen bonding in a doublestranded nucleic acid. The following base pairs are complementary:guanine and cytosine; adenine and thymine; and adenine and uracil.

The gene that is integrated by homologous recombination in accordancewith the present invention is a T7 RNA polymerase gene. The T7 RNApolymerase gene is obtained from T7 bacteriophage and is under controlof an isopropylthio-β-galactoside (IPTG) inducible lacUV5 promoter. Thegene can be obtained from plasmid pAR1219, American Type CultureCollection (ATCC) 39563, U.S. Pat. No. 4,952,496. A BamHI fragment inpAR1219 contains a T7 expression cassette comprising a T7 RNA polymerasegene under control of the IPTG-inducible lacUV5 promoter, and a lacIgene under control of the its native promoter.

The T7 RNA polymerase gene encodes a T7 RNA polymerase that is wellknown in the art and is described in detail in U.S. Pat. No. 4,952,496,which is herein incorporated by reference. When the host cell isinduced, a host cell RNA polymerase is able to bind to the lacUV5promoter and initiate synthesis of the T7 RNA polymerase.

“Nonintegrated gene” refers to a gene that is not integrated into thechromosome of a host cell, but is carried in an expression plasmid. Theexpression plasmid is introduced into the host cell by routine andconventional transformation methods, and replicates autonomously withinthe host cell at permissive temperatures. Thus, the plasmid canreplicate itself in the host cell in the absence of host cellduplication. The nonintegrated gene that is carried in the expressionplasmid encodes a target protein of interest. The nonintegrated gene isunder control of an isopropylthio-β-galactoside (IFFG) inducible T7 orT7lac promoter. The newly synthesized T7 RNA polymerase from theintegrated gene is able to bind to the T7 or T7lac promoter and initiatesynthesis of the target protein.

“Target protein” refers to a protein that can be synthesized in a hostcell. Preferably the target protein is heterologous to host cellproteins. Examples of proteins include but are not limited tocalcitonin, erythropoietin (EPO), factor IX, factor VIII, granulocytecolony stimulating factor (G-CSF), granulocyte macrophage colonystimulating factor (GM-CSF), macrophage colony stimulating factor(M-CSF), chemokines, growth hormone releasing factor (GRF), insulin-likegrowth factor (IGF-1), growth hormone, insulin, leptin, interferon,interleukins, luteinizing hormone releasing hormone (LHRH), folliclestimulating hormone (FSH), somatostatin, vasopressin, amylin,glucagon-like-peptide-1 (GLP-1), parathyroid hormone (PTH), exendin-3,exendin4, and alpha-1 anti-trypsin. The target protein of the presentinvention can optionally be a precursor protein or pro-protein. Examplesof precursor proteins or pro-proteins include but are not limited toproinsulin and GIP-1(1-37).

Integration Construct:

Useful plasmids are constructed to allow the integration of therecombinant target gene into the chromosome of a desired host cell byhomologous recombination. This integration can be accomplished usingmodified pMAK constructs. Preferably the starting pMAK constructs arepMAK700 and pMAK705. More preferably the starting pMAK construct ispMAK705. pMAK constructs comprise a temperature sensitive origin ofreplication. This allows the construct to replicate at permissivetemperatures like 30° C., but the construct will not replicate atnonpermissive temperatures like 44° C. The pMAK constructs also comprisea chloramphenicol resistance (Cm^(r)) gene. Thus, a host cell thatcontains a plasmid that comprises a Cm^(r) gene will be resistant tochlorampehicol, and at a permissive temperature will replicate in thepresence of chloramphenicol.

The pMAK constructs are modified by the insertion of nucleic acidsequences into the pMAK construct that are homologous to a nucleic acidsequence found on the chromosome of a host cell. pMAK constructs whichare inserted with homologous nucleic acid sequences found on thechromosome of a host cell are referred to in the present invention aspHMM constructs. The homologous sequences of the pHMM constructscomprise different fragments of the galactose operon (galETK). Thegalactose operon is well known in the art. The homologous sequences ofthe pHMM construct and the host cell have sufficient length to hybridizeto each other and undergo recombination. The hybridization generallydepends on the ability of denatured chromosomal DNA to re-anneal whencomplementary strands from the integration construct are present in anenvironment like a host cell. Preferably the homologous sequence isgreater than about 1 kb. More preferably, the homologous sequence isbetween about 1 kb and about 10 kb. Even more preferably, the homologoussequence is between about 1 kb and about 4 kb. Most preferably, thehomologous sequence is about 2 kb. The homologous sequences of the hostcell can be any sequence that is not essential to the host cell becausethe recombination event can disrupt the sequence such that the sequencecan become nonuseful. For example, if the homologous sequence is in thegene responsible for the synthesis of the cell wall, recombination inthis sequence of the host cell with the integration plasmid coulddisrupt synthesis of the proteins comprising the cell wall and result ina nonviable host cell.

The pHMM constructs can be further modified by the insertion of a T7 RNApolymerase gene and a lacUV5 promoter into the pHMM construct. A T7 RNApolymerase gene under control of the lacUV5 promoter can be obtainedfrom plasmid pAR1219, American Type Culture Collection (ATCC) 39563,U.S. Pat. No. 4,952,496.

Preferably, the original lac promoter of the pMAK plasmid is eliminatedby the cloning of the T7 RNA polymerase gene and the lacUV5 promoterinto the pHMM construct. A duplication of lac promoters could result inthe potential for secondary structure formation, which might presentproblems for sequence determination and the possibility for interferingwith homologous recombination.

Optionally, a pHMM construct can be further modified by the insertion ofa lacI gene into the pHMM construct. In addition to the T7 RNApolymerase gene and the lacUV5 promoter, the pAR1219 plasmid furthercomprises a DNA fragment containing the lacI gene under control of itsnative promoter. A copy of the lacI gene in the expression system canprovide additional expression of the lactose repressor which helpscontrol both T7 RNA polymerase and target protein expression.

Optionally, the lacI gene from the T7 expression cassette driven by alacI^(Q1) promoter. The lacI^(Q1) promoter is well known in the art. ThelacI^(Q1) promoter is modified to overexpress the lad gene. The resultis about 100× production of the lacI repressor than the lacI gene drivenby its native promoter.

Additionally, a pHMM construct further comprises a second resistancegene. Preferably the second resistance gene is kanamycin (Km^(r)). Thus,a host cell that contains a plasmid that comprises a Km^(r) gene will beresistant to kanamycin and will replicate in the presence of kanamycin.Preferably, the second resistance gene is oriented in the oppositedirection as the T7 RNA polymerase. The kanamycin resistance geneprovides an additional means of uniquely identifying the host cell. Thekanamycin resistance gene can be obtained from the plasmid pACYC177.pACYC177 is available from “Stratagene Cloning Systems” Catalog (1993)(Stratagene, La Jolla, Calif.). The kanamycin resistance gene frompACYC177 includes Tn903 transposition inverted repeats (IR). Due topotential instability through transposition resulting from the presenceof these inverted repeats, a cassette encompassing the kanamycinresistance gene but not the inverted repeat sequences is preferred.

Integration:

An integration construct can be transformed into a desired host strainaccording to conventional methods and individual colonies are grownovernight in liquid growth media at permissive temperature in thepresence of a selection agent, for example, Cm or Km. The resultingovernight culture is diluted in liquid growth media in the presence of aselecting agent and incubated at a nonpermissive temperature, forexample, 44° C., until log phase. The culture is then plated on agarplates containing a selection agent and incubated overnight atnonpermissive temperature to select for cointegrate formation.Cointegrate formation is the initial step in homologous recombinationand occurs when the integration construct integrates into the hostchromosome. Because the integration plasmid cannot replicate itself at anonpermissive temperature and the culture contains a selecting agent,the only host cells that will survive under these conditions will bethose that integrate the integration construct into the host cellchromosome. The resulting culture is plated on agar plates comprising aselecting agent and incubated at nonpermissive temperature overnight toselect for cointegrates.

A pool of cointegrate colonies are picked, transferred to liquid growthmedia, and incubated overnight at permissive temperature for resolutionof the cointegrate. Resolution provides a means for a secondrecombination event to occur whereby the integration plasmid is excisedfrom the chromosome and reformed within the host cell. The integrationplasmid that is excised and reformed in the host cell is either theoriginal integration plasmid in whole or is the original integrationplasmid minus the T7 RNA polymerase which remains integrated into thechromosome of the host cell. The objective of the second recombinationevent is to excise the portion of the integration plasmid that comprisesthe origin of replication from the host cell chromosome, but to leavethe T7 RNA polymerase integrated into the chromosome of the host cell. Aschematic of this process is shown in FIG. 1. In the cases where theintegration plasmid further comprises other genes, for example lacI orKm, the objective of the second recombination event is to excise theportion of the integration plasmid that comprises the origin ofreplication from the host cell chromosome, but to leave the T7 RNApolymerase, and other genes, for example lacI or Km, integrated into thechromosome of the host cell. Removal of the origin of replication of theintegration plasmid is desired because an integrated origin ofreplication could be deleterious to the host cell. This excision processmay optionally be continued for days by subculturing with a selectingagent and maintaining at permissive temperature. Preferably,subculturing and maintaining is less than three days, more preferablysubculturing and maintaining is continued for two days.

The culture is then diluted into a pre-warmed flask contain liquidgrowth media without a selecting agent at nonpermissive temperature toinitiate curing of the integrate by excising undesirable plasmidsequence from the chromosome of the host cell. The culture is plated onagar plates containing a selection agent and grown at permissivetemperature. Colonies are screened for the presence of an integrationevent using means known to a skilled artisan, for example, PCR andSouthern blotting. Colonies containing an integrate are used toinoculate a liquid media culture and subsequently grown for consecutivedays at nonpermissive temperature to promote curing. The cultures andcan then plated onto agar plates and incubated overnight at permissivetemperature. Individual colonies can subsequently be patched onto agarplates optionally containing both selecting agents, for example Cm andKm. The individual colonies can further be patched onto agar platescontaining only the second selecting agent, for example Km. The desiredclones which have integrated sequences are Cm sensitive and Kmresistant.

In another embodiment, the integration plasmid is preferably integratedinto the galactose operon of the host cell. More preferably, theintegration plasmid is integrated into the galE locus of the host cell.Several attempts were made to integrate into the galK locus, howeverideal integration was unsuccessful.

Target Protein:

The nonintegrated gene encoding a recombinant target protein used in theexpression system of the present invention is obtained by meansavailable to ordinarily skilled artisans in the field of molecularbiology. The basic steps are:

-   -   a) isolating a natural DNA sequence or constructing a synthetic        or semi-synthetic DNA sequence, wherein either DNA sequence        comprises a target gene that encodes a target protein of        interest,    -   b) cloning the DNA sequence into an available T7 expression        plasmid in a manner suitable for expressing the target protein,    -   c) transforming the previously described expression host of the        present invention with the T7 expression plasmid comprising the        target gene of interest,    -   d) culturing the transformed expression host for a period of        time in an uninduced state and then for a period of time in an        induced state, and    -   e) recovering and purifying the target protein.

Preferably, the target protein is parathyroid hormone (PTH). Morepreferably, the PTH is human PTH. PTH is known in the art as an 84 aminoacid protein and described in U.S. Pat. No. 5,496,801. N-terminalfragments of PTH are also well known in the art and include but are notlimited to 1-34, 1-31, and 1-28. Also, contemplated are analogs andderivatives of PTH and PITH fragments. Examples of PTH fragments,analogs and derivatives are described in WO99/29337, U.S. Ser. No.20020132973, U.S. Pat. Nos. 5,556,940; 6,472,505; and 6,417,333.

In another embodiment, the target protein is glucagon-like peptide-1(GLP-1), or analogs or derivatives thereof. Examples of GLP-1 analogsand derivatives are well known in the art and are described inWO01/98331, and U.S. Pat. Nos. 6,268,343; 5,977,071; 5,545,618;5,705,483; and 6,133,235. GLP-1 analogs also include Exendin-3 andExendin-4 agonists as described in WO99/07404, WO99/25727, WO99125728,WO99/43708, WO00/66629, and U.S. Ser. No. 2001/0047084A1.

Modification:

The isolated target protein is useful as a therapeutic protein.Optionally the target protein can be further modified outside the hostcell to give the target protein additional physical characteristicsuseful for a therapeutic protein. Modifications include but are notlimited to enzymatic or chemical cleavages, acylation, crystallization,salt additions, and the like.

Prepartations:

Liquid growth media is T Broth

T Broth=(per liter) 10 g tryptone, 5 g yeast extract, 10 g NaCl, pH 7.5.

T agar plates=add 15 g/L agar to T broth.

SM buffer=(per 100 mL of 10× solution) 20 mL 1M Tris-HCl (pH 7.4), 20 mL5M NaCl, 10 mL 1M MgSO₄

Chloramphenicol (Cm)(25 ug/mL) in ethanol.

Kanamycin (Kam)(15-50 ug/mL) in water.

Nalidixic acid (20 ug/mL) in NaOH

Streptomycin (50 ug/mL) in water

Integration Plasmid pHMM209:

The integration plasmid pHMM209 is a pMAK705 derivative. The initialstep in the construction of pHMM209 is to clone an oligonucleotideadapter, BamHI to ClaI, into the pMAK705 backbone. This adapter containsa StuI site, which is unique in the resulting construct. A galK flank iscloned into the pMAK705 backbone as a SalI to XbaI insert resulting in apHMM backbone. The pHMM backbone comprises unique BamHI and ClaI sitesin the galK flank. The T7 expression cassette from pAR1219, comprisingthe lacI gene under the expression of its native promoter sequence andthe T7 RNA polymerase gene under the regulation of the lacUV5 promoteris then cloned as a BamHI fragment into the pHMM backbone. Theorientation of the T7 expression cassette is opposite that of the galETKoperon to prevent transcriptional read-through from galE upstreamsequences. Next, the resistance gene for kanamycin is cloned as a StuIfragment from pACYC177 into the StuI site of the adapter that waspreviously cloned into the pMAK705 backbone. The orientation of thekanamycin gene is opposite that of the T7 expression cassette. Theresulting integration plasmid is pHMM209.

Integration Plasmid pHMM220:

The integration plasmid pHMM220 is a pMAK705 derivative. The initialstep in the construction of pHMM220 is to clone an oligonucleotideadapter, BamHI to ClaI, into the pMAK705 backbone. This adapter containsa StuI site, which is unique in the resulting construct. A galK flank iscloned into the pMAK705 backbone as a SalI to XbaI insert resulting in apHMM backbone. The pHMM backbone comprises in unique BamHI and ClaIsites in the galK flank. The T7 expression cassette, comprising the lacIgene under the expression of its native promoter sequence and the T7 RNApolymerase gene under the regulation of the lacUV5 promoter is thencloned as a BamHI fragment from pAR1219 into the pHMM backbone. Theorientation of the T7 expression cassette is opposite that of the galETKoperon to prevent transcriptional read-through from galE upstreamsequences. Next, a kanamycin resistance gene as a StuI fragment isobtained by PCR. The PCR primers that are used to amplify the resistancegene are designed inside the inverted repeat sequences present in thepACYC177 template kanamycin gene. The PCR primers contain StuIrestriction sites in their tails and they are used in an amplificationreaction. The resulting approximately 1 kb PCR product is cloneddirectly into a PCR cloning plasmid and putative clones are selected forby plating directly on T agar plates containing kanamycin. The resultingkanamycin resistance gene is subcloned as a StuI fragment into the StuIsite of the adapter that was previously cloned into the pMAK705backbone. The orientation of the kanamycin gene is opposite that of theT7 expression cassette. The resulting integration plasmid is pHMM220.

Integration Plasmid pHMM223:

Integration plasmid pHMM223 is constructed the same as pHMM220. Next,the lad gene of the T7 expression cassette in pHMM220 is removed becausethe lacI gene had the potential to integrate into the lacI locus of thehost chromosome. The lacI gene is deleted from the pHMM220 by digestionof the plasmid using BglI. A synthetic DNA adapter is cloned into theBglI site to reconstitute the lacUV5 promoter that is deleted in theBglI digestion process. The resulting clone is sequenced and is found tocontain the desired lacUV5 sequence with the exception of two nucleotidechanges. These changes are in the 5′ untranslated region of the T7expression cassette and are not critical to expression of the T7 RNApolymerase. Next, the lacI promoter present in the pHMM220 iseliminated. This is accomplished by insertion of a PstI to AseI adapterthat completely replaces the lacI promoter sequence.

The BglI deletion of the pHMM220 also removes the downstream galK flank.In order to reconstitute this region and incorporate the kanamycinresistance gene without the inverted repeats, a BamHI to XbaI fragmentis subcloned into the BglII to XbaI sites of the integration plasmid.This results in an integration plasmid designated pHMM223, whichcontains the T7 expression cassette without a copy of the lacI gene andthe lacI promoter, kanamycin resistance gene without inverted repeats,and a complete galK flank. The pHMM223 is used for attempts to integrateinto the galK locus of the chromosome.

Integration Plasmid pHMM228:

The integration plasmid pHMM228 is a pMAK705 derivative. The initialstep in the construction of pHMM228 is to clone an oligonucleotideadapter, PstI to EagI, into the pMAK705 backbone. This adapter containsunique SalI and XbaI sites. Approximately 2 kb of the galE gene iscloned into the pMAK705 backbone as a SalI to XbaI insert resulting in apHMM backbone. The pHMM backbone comprises unique BamHI and ClaI sitesin the gene. The T7 expression cassette, comprising the lacI gene underthe expression of its native promoter sequence and the T7 RNA polymerasegene under the regulation of the lacUV5 promoter is then cloned as aBamHI fragment from pAR1219 into the pHMM backbone. The orientation ofthe T7 expression cassette is opposite that of the galETK operon toprevent transcriptional read-through from galE upstream sequences. Next,a kanamycin resistance gene as a StuI fragment is obtained by PCR. ThePCR primers that are used to amplify the resistance gene are designedinside the inverted repeat sequences present in the pACYC177 templatekanamycin gene. The PCR primers contain StuI restriction sites in theirtails and they are used in an amplification reaction. The resultingapproximately 1 kb PCR product is cloned directly into a PCR cloningplasmid and putative clones are selected for by plating directly on Tagar plates containing kanamycin. The resulting kanamycin resistancegene is subcloned as a StuI fragment into the StuI site of the adapterthat was previously cloned into the pMAK705 backbone. The orientation ofthe kanamycin gene is opposite that of the T7 expression cassette.Finally, the lacI gene of the T7 expression cassette is removedessentially as described for pHMM223. The lacI gene is deleted bydigestion of the plasmid using BglI. A synthetic DNA adapter is clonedinto the BglI site to reconstitute the lacUV5 promoter that is deletedin the BglI digestion process. Next, the lacI promoter is eliminated byinsertion of a PstI to AseI adapter that completely replaces the lacIpromoter sequence. The BglI deletion also removes the downstream galEflank. In order to reconstitute this region and incorporate thekanamycin resistance gene without the inverted repeats, a BamHI to XbaIfragment is subcloned into the BglII to XbaI sites of the integrationplasmid. This results in an integration plasmid designated pHMM228,which contains the T7 expression cassette without a copy of the lacIgene and lacI promoter, kanamycin resistance gene without invertedrepeats, and a complete galE flank. The pHMM228 is used for attempts tointegrate into the galE locus of the chromosome.

Integration/Screening of pHMM209:

The integration plasmid pHMM209 is transformed into a E. coli parentalcell line comprising a galactose operon, plated on T agar platescontaining Cm, and incubated overnight at 30° C. Colonies where picked,transferred to T broth containing Cm and grown overnight at 30° C. Theresulting overnight culture is diluted in T broth in the presence of Cmand incubated at 44° C., until the culture reaches log phase. Theculture is then plated on T agar plates comprising Cm and incubatedovernight at 44° C. to induce cointegrate formation. A pool ofcointegrate colonies are picked, transferred to 250 mL of T brothcontaining Cm, and incubated overnight at 30° C. for excision andresolution. This culture is maintained for two more days bysub-culturing at a 1:500 dilution with T broth containing Cm andincubating the flask at 30° C. On the fourth day, the culture issub-cultured into a pre-warmed flask of T broth at 44° C. This cultureis grown and sub-cultured for three consecutive days at 44° C. topromote curing of the pHMM209 plasmid. The tentatively integrated,excised and cured culture is then plated onto T agar plates containingKm and incubated overnight at 30° C. Individual colonies aresubsequently patched onto T agar plates containing Cm and Km, then ontoT agar plates containing Km, then onto T agar plates. Positiveintegrates should be Cm^(s) and Km^(r).

Nearly 1000 individual colonies were tested and only one integrate wasformed. This integrate is designated RQ209. Further analyses showed thatthe RQ209 strain possessed functional T7 RNA polymerase that was inducedby the addition of IPTG. However, when PCR mapping was performed on theRQ209 strain, it was found that the T7 RNA polymerase had notspecifically integrated into the galK or the lacI regions of thechromosome.

Integration/Screening for pHMM228:

Integration experiments were carried out essential as described inintegration/screening of pHMM209 above. The table below shows the numberof cointegrates formed. TABLE 1 Cointegrates of pHMM228 Plate Counts10⁻¹ 10⁻² 10⁻³ 10⁻⁴ 10⁻⁵ 10⁻⁶ 10⁻⁷ Plating ND ND ND TNTC 73 8 1Temperature 30° C. Plating TNTC TNTC TNTC 153 11 0 ND Temperature 44° C.TNTC: to numerous to countND: not determined

Colonies that grew on the 44° C. plates were subsequently grown in Tbroth at 44° C. Nine individual isolates were grown in addition to oneculture that was pooled from colonies representing approximately ½ anentire plate. These 10 cultures were shaken (315 rpm) overnight under Cmselection at 44° C. The following day 100 uL samples from each wereharvested by centrifugation for subsequent PCR analyses. In addition,plates pre-warmed to 44° C. were used to streak for individual isolatesfrom these cultures and incubated overnight at 44° C. PCR andrestriction mapping results showed that nearly all of the 10 liquidcultures contained an amplification product consistent with the expectedintegration event.

The individuals clones were screened by re-patching at 44° C. Individualisolate #2 was grown up in T broth containing Cm at 30° C. overnight topromote excision of pHMM228. After overnight growth, a 100 uL sample ofcells were collected and used as template for a PCR reaction. Primersfrom outside the galE flanks were chosen so that the only amplificationwould be the excised version of pHMM228. Thus if the excision eventregenerates pHMM228, an approximately 7 kb PCR product would beexpected. However, if excision resulted from a second crossover eventleaving the T7 RNA polymerase in the chromosome, an approximately 1.5 KbPCR product would be expected. As expected, a mixture of excisionproducts is observed. The excised culture is subsequently streaked outto obtain individual isolates which are screened for the presence of the1.5 Kb PCR product.

Three isolates were then grown overnight without selection at 44° C. inT broth to promote curing of the excised pHMM228. After overnight growthat 44° C., single colonies were isolated from streaked T agar plates and72 individuals from each of the three original isolates were patchedonto T plates plus Cm, T plates plus Km, and T plates to determine thosethat had been successfully cured of the excised pHMM228. The table belowdetails the results of these experiments. TABLE 2 Curing EfficiencyTotal Individuals Curing Isolate Analyzed Cm^(r) Km^(r) Efficiency (%)#1 72 24 72 66.7 #6 72 37 72 48.6 #15 72 14 56 56.9

A single Cm-sensitive individual designated RQ228 was subsequentlychosen from the isolate #1 and was streaked for purification two timesand phenotypically verified. The table below shows the results of thephenotypic analyses. TABLE 3 Phenotypic Results Result of PhenotypePlate Patching M9 no growth M9 + galactose no growth M9 + lactose growthM9 + glucose growth L + Cm no growth L + streptomycin growth L + Kmgrowth L + Nalidixic Acid no growth L growth

A colony of the RQ228 strain that was phenotype confirmed was thenchosen and a 10 mL culture was grown up overnight for local as well aslong-term preservation and used to make a competent cell lot. This samecolony was used in integration integrity PCR mapping.

T7 Activity and Regulation Assay:

In addition to confirming the phenotypic characteristics and theintegrity of the integration event, the RQ228 strain was also examinedfor its ability to express functional T7 RNA polymerase as well as theability of this expression to be regulated. The ability of the RQ228strain to rescue the defective T7 tester phage was examined as describedbelow.

T7 RNA Polymerase Assay:

A T7 RNA polymerase activity assay was utilized in order to determinewhether the RQ209 strain or the RQ228 strain possessed functional T7 RNApolymerase. The RQ209 strain or the RQ228 strain were grown at about 37°C. overnight in T broth supplemented with 0.2% maltose and 10 mM MgSO₄.Overnight cultures were diluted back to OD₆₀₀=0.05 in T broth againsupplemented with 0.2% maltose and 10 mM MgSO₄ and grown shaking toOD₆₀₀=0.5 and 100 uL of each bacterial culture was added to 100 uL of a10⁻⁶ dilution in SM buffer of the T7 tester phage. The samples weregently mixed by finger vortexing and incubated at 37° C. for 20 minutesto allow phage adsorption. Three mL of 0.4% T top agarose (supplementedwith 0.2% maltose and 10 mM MgSO₄) was then added to the samples,vortexed and poured onto pre-warmed T agar plates. Each sample wasprepared in duplicate so that one could be plated on T agar and theother could be plated on T agar containing 400 uM IPTG. The T7 testerphage can attach to the cells but can replicate and lyse only thosecells it infects that have functional T7 RNA polymerase available. SinceT7 RNA polymerase expression is under the control of the lacUV5promoter, expression should only result in presence of the inducer,IPTG. Plaques on the plate containing EPTG and no plaques on the platewithout IPTG constitutes a positive indication of controlled expressionof T7 RNA polymerase.

Final PCR Confirmation of Integration Integrity:

The RQ228 strain was analyzed to confirm the integrity/specificity ofthe integration event. PCR amplifications were performed to examine bothjunctions of the galE-targeted integration event as well as confirmationof the size of the entire integration cassette.

PTH Expression Using RQ228:

An expression plasmid comprising the gene for PTH is transformed intoeither of RQ228 or a DE3 host cell using conventional methods. Bothstrains are grown at 37° C. in T broth containing tetracycline andinduced by the addition of IPTG to 10 uM. The cultures are continued toincubate for 6 hours. Samples of the cultures show that both host cellsexpress PTH. However, only the PTH produced in the RQ228 host cell isphage-free, while the PTH produced in the DE3 host cell has measurablelevels of phage contamination in the fermentation broth.

E. coli is grown to saturation at 37° C. overnight in T brothsupplemented with 0.2% maltose and 10 mM MgSO₄. The overnight culture isdiluted back to OD₆₀₀=0.05 in T broth again supplemented with 0.2%maltose and 10 mM MgSO₄ and grown shaking to. OD₆₀₀=0.5 and 100 uL of E.coli culture is added to 100 uL of fermentation broth. The sample isgently mixed by finger vortexing and incubated at 37° C. for 20 minutesto allow phage adsorption. Three mL of 0.4% T top agarose (supplementedwith 0.2% maltose and 10 mM MgSO₄) is added to the sample, vortexed andpoured onto pre-warmed T agar plates. The plates are incubated at 37° C.for about 12 hours. Phage-free fermentation broth will produce noobservable plaques.

1. A host cell comprising a homologously integrated T7 RNA polymerasegene under control of a lac promoter.
 2. The host cell of claim 1wherein the T7 RNA polymerase is integrated into a host cell chromosomewithout the use of a phage lysogen.
 3. The host cell of claim 2 whereinthe lac promoter is lacUV5 promoter.
 4. The host cell of claim 2 or 3wherein the T7 RNA polymerase gene is integrated into the galactoseoperon of the host chromosome.
 5. The host cell of claim 4 wherein theT7 RNA polymerase gene is integrated into the galactose operon from anintegration plasmid selected from the group consisting of pHMM209,pHMM22, pHMM223 and pHMM228.
 6. The host cell of any one of claims 2 to5 wherein the host cell further comprises a nonintegrated gene encodinga target protein under control of a T7lac promoter.
 7. The host cell ofclaim 6 wherein the target protein is parathyroid hormone (PTH).
 8. Thehost cell of claim 7 wherein the PTH is an N-terminal fragment of 1-84.9. The host cell of claim 8 wherein the N-terminal fragment is 1-34. 10.The host cell of claim 6 wherein the target protein is glucagon-likepeptide-1 (GLP-1), or a GLP-1 analog or derivative.
 11. An expressionsystem for producing a target protein in phage-free fermentation broth,wherein the expression system comprises a host cell with a homologouslyintegrated T7 RNA polymerase gene in a nonessential gene of a host celland a nonintegrated gene encoding the target protein, and wherein thenonintegrated gene is under control of a T7lac promoter.
 12. Theexpression system of claim 11 wherein the T7 RNA polymerase gene isintegrated into the galactose operon of the host chromosome.
 13. Theexpression system of claim 12 wherein the T7 RNA polymerase gene isintegrated into the galactose operon from an integration plasmidselected from the group consisting of pHMM209, pHMM22, pHMM223 andpHMM228.
 14. The expression system of claim 13 wherein the targetprotein is parathyroid hormone (PTH).
 15. The expression system of claim14 wherein the PTH is an N-terminal fragment of 1-84.
 16. The expressionsystem of claim 15 wherein the N-terminal fragment is 1-34.
 17. Theexpression system of claim 13 wherein the target protein isglucagon-like peptide-1 (GLP-1), or a GLP-1 analog or derivative.
 18. Aprocess of preparing a host cell comprising homologously integrating aT7 RNA polymerase gene under control of a lacUV5 promoter into anonessential gene of the host, such that upon induction of the T7 RNApolymerase gene the fermentation broth will be phage-free.
 19. Theprocess of claim 18 wherein the T7 RNA polymerase gene is integratedinto the galactose operon.
 20. The process of claim 19 wherein the T7RNA polymerase gene is integrated into the galactose operon from anintegration plasmid selected from the group consisting of pHMM209,pHMM22, pHMM223 and pHMM228.
 21. A process for preparing a targetprotein which comprises a) preparing a host cell comprising homologouslyintegrating a T7 RNA polymerase gene under control of a lacUV5 promoterinto a nonessential gene of the host, b) transforming the host cell witha nonintegrated gene encoding a target protein, and wherein thenonintegrated gene is under control of a T7lac promoter, c) inducing thehost cell to produce T7 RNA polymerase, d) incubating the host cell infermentation broth for a time sufficient to allow the T7 RNA polymeraseto produce the target protein, and wherein the fermentation broth willbe phage-free